Effects of noise on the performance of rats in an operant discrimination task

Two experiments examined the effect of the presentation of an irregular, moderate intensity auditory stimulus ('noise') on the performance of rats in an operant discrimination task. In Experiment 1, rats first learned to press a lever in the presence of a visual stimulus but not in its absence. Discrimination performance was impaired during subsequent exposure to noise. In Experiment 2, different groups of rats learned the discrimination task under a noise or a no-noise condition. Thereafter, all rats were tested under each noise condition. Discrimination performance was best when the noise condition at test was identical to the noise condition at training. These results were discussed in the framework of arousal, distraction, generalization decrement, and contextual occasion setting. They point to the necessity of using a 2x2 factorial design in human and animal research on noise effects, with noise condition at training (noise present or absent) and noise condition at test (noise present or absent) as factors.     

The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor

Antimicrobial peptides produced by epithelial cells and neutrophils represent essential elements of innate immunity, and include the defensin and cathelicidin family of antimicrobial polypeptides. The human cathelicidin cationic antimicrobial protein-18 is an antimicrobial peptide precursor predominantly expressed in neutrophils, and its active peptide LL-37 is released from the precursor through the action of neutrophil serine proteinases. LL-37 has been shown to display antimicrobial activity against a broad spectrum of microorganisms, to neutralize LPS bioactivity, and to chemoattract neutrophils, monocytes, mast cells, and T cells. In this study we show that LL-37 activates airway epithelial cells as demonstrated by activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and increased release of IL-8. Epithelial cell activation was inhibited by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, by the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478, by blocking anti-EGFR and anti-EGFR-ligand Abs, and by the metalloproteinase inhibitor GM6001. These data suggest that LL-37 transactivates the EGFR via metalloproteinase-mediated cleavage of membrane-anchored EGFR-ligands. LL-37 may thus constitute one of the mediators by which neutrophils regulate epithelial cell activity in the lung.     

Immobilization of the distal hinge in the labile serpin plasminogen activator inhibitor 1: identification of a transition state with distinct conformational and functional properties

The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.     

p116Rip is a novel filamentous actin-binding protein

p116Rip is a ubiquitously expressed protein that was originally identified as a putative binding partner of RhoA in a yeast two-hybrid screen. Overexpression of p116Rip in neuroblastoma cells inhibits RhoA-mediated cell contraction induced by lysophosphatidic acid (LPA); so far, however, the function of p116Rip is unknown. Here we report that p116Rip localizes to filamentous actin (F-actin)-rich structures, including stress fibers and cortical microfilaments, in both serum-deprived and LPA-stimulated cells, with the N terminus (residues 1-382) dictating cytoskeletal localization. In addition, p116Rip is found in the nucleus. Direct interaction or colocalization with RhoA was not detected. We find that p116Rip binds tightly to F-actin (Kd approximately 0.5 microm) via its N-terminal region, while immunoprecipitation assays show that p116Rip is complexed to both F-actin and myosin-II. Purified p116Rip and the F-actin-binding region can bundle F-actin in vitro, as shown by electron microscopy. When overexpressed in NIH3T3 cells, p116Rip disrupts stress fibers and promotes formation of dendrite-like extensions through its N-terminal actin-binding domain; furthermore, overexpressed p116Rip inhibits growth factor-induced lamellipodia formation. Our results indicate that p116Rip is an F-actin-binding protein with in vitro bundling activity and in vivo capability of disassembling the actomyosin-based cytoskeleton.     

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties

Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M. kansasii pathogenicity. Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response. Therefore, the detailed structures of the of M. kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient. Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species. KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis. Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM. With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM. Because M. kansasii has been reported to induce apoptosis, we examined the possibility of the M. kansasii lipoglycans to induce apoptosis of THP-1 cells. Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor. This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M. kansasii infections by inducing apoptosis.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

Bacillus subtilis hydrolyzes CheY-P at the location of its action,the flagellar switch

In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P to form CheY and Pi. This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch. In particular we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC, a protein found in the archaea and many bacteria but not in E. coli. Previous E. coli studies have localized the CheY-P binding site of the switch to FliM residues 6-15. These residues are almost identical to the residues 6-15 in both B. subtilis FliM and FliY. We were able to show that both of these proteins are capable of binding CheY-P in vitro. Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite. We showed that FliY increases the rate of CheY-P hydrolysis in vitro. Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.     

Evidence of regio-specific glycosylation in human intestinal mucins: presence of an acidic gradient along the intestinal tract

Mucin glycans were isolated from different regions of the normal human intestine (ileum, cecum, transverse and sigmoid colon, and rectum) of two individuals with ALeb blood group. A systematic study of the monosaccharides and oligosaccharide alditols released by reductive beta-elimination from mucins was performed using gas chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy techniques. Important variations were observed in the mucin-associated oligosaccharide content with an increasing gradient of sialic acid from the ileum to the colon associated with a reverse gradient of fucose. Moreover, a comparative study of the Sda/Cad and ABH blood group determinants along the gastrointestinal tract showed the same reverse distribution in the two kinds of antigens. In addition, besides their heterogeneity, sialic acids presented considerable variations in the degree of O-acetylation in relation to glycan sialylation level. These data are discussed in view of recent concepts suggesting that the oligosaccharide composition of the gut constitutes a varied ecosystem for microorganisms that are susceptible to adapt there and possess the specific adhesion system and specific enzymes able to provide a carbohydrate nutrient.     

Embryonic cell lineage of the marine nematode Pellioditis marina

We describe the complete embryonic cell lineage of the marine nematode Pellioditis marina (Rhabditidae) up to somatic muscle contraction, resulting in the formation of 638 cells, of which 67 undergo programmed cell death. In comparison with Caenorhabditis elegans, the overall lineage homology is 95.5%; fate homology, however, is only 76.4%. The majority of the differences in fate homology concern nervous, epidermal, and pharyngeal tissues. Gut and, remarkably, somatic muscle is highly conserved in number and position. Partial lineage data from the slower developing Halicephalobus sp. (Panagrolaimidae) reveal a lineage largely, but not exclusively, built up of monoclonal sublineage blocs with identical fates, unlike the polyclonal fate distribution in C. elegans and P. marina. The fate distribution pattern in a cell lineage could be a compromise between minimizing the number of specification events by monoclonal specification and minimizing the need for migrations by forming the cells close at their final position. The latter could contribute to a faster embryonic development. These results reveal that there is more than one way to build a nematode.